Immunostimulatory composition and use thereof

ABSTRACT

Provided is an immunostimulatory composition, comprising a saponin and a CpG oligodeoxynucleotide, or consisting of an adjuvant comprising a saponin and a CpG oligodeoxynucleotide, wherein the sequence of the CpG oligodeoxynucleotide has two or more copies of 5′-TTCGTT-3′ motif or 5′-TCGTCGTCG-3′ motif. Also provided is use of the immunostimulatory composition in the preparation of a medication for treating diseases.

TECHNICAL FIELD

The present invention belongs to the field of biopharmaceutics. Inparticular, the present invention relates to an immunostimulatorycomposition comprising a saponin and a CpG oligodeoxynucleotide, orconsisting of an adjuvant comprising a saponin and a CpGoligodeoxynucleotide, wherein the sequence of the CpGoligodeoxynucleotide has two or more copies of 5′-TTCGTT-3′ motif or5′-TCGTCGTCG-3′ motif. The present invention also relates to use of theimmunostimulatory composition in the manufacture of a medicament.

BACKGROUND ART

CpG oligodeoxynucleotides are a new class of immunostimulatory agentsdiscovered in recent years, and their chemical nature is anoligodeoxynucleotide containing cytosine-guanine dinucleotide, whichhave a similar immune response to the natural pattern recognitionreceptors for CpG, and can bind to Toll-like receptors on cell membrane,effectively triggering a mammalian immune response through TLR9signaling pathway. The immunoreaction triggered by CpG is mainly ofTh1-type, which can induce an immune response conversion from Th2-typeto Th1-type, thus stimulating cellular immunity. Activatingimmunoreactive cells, such as T cells, B cells, and NK cells, etc. cangenerate a large amount of multiple cytokines, thereby enhancing thespecific and non-specific immune effects in the body, which is animportant link between natural immunity and acquired immunity.

Saponins are a class of glycosides, the aglycons of which are triterpeneor spirostane compounds, and belong to plant-derived adjuvants. Amongthem, quillaja saponin (QS) is the saponins extracted from quillaja, andQS-21 is the most widely reported adjuvant in QS series. However, QS-21may induce cell hemolysis and has some systemic and local toxic/sideeffects. Alving et al. study (ALVING CR, MATYAS G, BECK Z, et al. RevueRoumaine de Chimie, 2016, 61(8): 631-635) found that ALF liposomes incombination with MPLA and QS-21 as an adjuvant against HIVgp140 proteincould effectively increase the antibody titer in serum. Ng et al. (NG H,FERNANDO G J P, DEPELSENAIRE A C I, et al. Scientific Reports, 2016,6(1): 228-230) used a subcutaneous delivery technique, a nano-patch, toform an adjuvant complex with QS-21. The results showed, compared withtraditional intramuscular injection, the nano-patch could significantlyreduce the dosages of antigen and QS-21, and induce a higher IgG titer(Ziyi Han, Zhongliang Zeng, Modern Agricultural Science and Technology,2019 (14): 220-221).

Immunostimulatory compositions comprising a saponin and a CpGoligodeoxynucleotide have been reported in the prior art(WO2001051083A3), wherein the CpG oligodeoxynucleotide involves CpG1826and CpG7909. However, the effects of CpG adjuvants having differentsequences differentiate greatly due to the structural diversity of CpGoligodeoxynucleotides.

Therefore, there is a current need for adjuvants and drugs with astronger immune effect.

CONTENTS OF THE INVENTION

In view of the deficiencies in the prior art, the inventors haveunexpectedly discovered, after extensive research, an immunostimulatorycomposition with a stronger immune effect. In the composition, thesaponin and the CpG oligodeoxynucleotide show a synergistic effect withhigh efficiency, which can mediate a more potent immune response. Theimmunostimulatory composition has significant advantages when applied todifferent antigens or antigen compositions.

Therefore, it is an object of the present invention to provide animmunostimulatory composition, which can be used to prepare variousdrugs to obtain immunostimulating immunogenicity with high efficiency.

It is another object of the present invention to provide a vaccineadjuvant which can strongly elicit an immune response in a mammal.

The objects of the invention are achieved by the following technicalsolutions.

In one aspect, the present invention provides an immunostimulatorycomposition comprising a saponin and a CpG oligodeoxynucleotide, orconsisting of an adjuvant comprising a saponin and a CpGoligodeoxynucleotide, wherein the sequence of the CpGoligodeoxynucleotide has two or more copies of 5′-TTCGTT-3′ motif or5′-TCGTCGTCG-3′ motif.

In the immunostimulatory composition according to the present invention,the sequence of the CpG oligodeoxynucleotide is any one selected from:CpG T1: TCG TTC GTT COT TCG TTC GTT (SEQ ID NO: 6); CpG T2: TCG TTC GTTCGT TCG TTC GTT CGT T (SEQ ID NO: 7); and CpG T3: TCG TCG TCG TCG TCGTCG TCG (SEQ ID NO: 8).

Preferably, the sequence of the CpG oligodeoxynucleotide is CpG T1: TCGTTC GTT CGT TCG TTC GTT (SEQ ID NO: 6).

In the immunostimulatory composition according to the present invention,the saponin is one or more selected from the group consisting ofquillaja saponin, ginsenoside, platycodin, astragaloside,notoginsenoside, glycyrrhizin, cortex albiziae saponin, ophiopogonin,saikosaponin or panax japonicus saponin. Preferably, the saponin isquillaja saponin, ginsenoside, platycodin or astragalin A. Morepreferably, the quillaja saponin is QS-7, QS-17, QS-18 or QS-21. Morepreferably, the quillaja saponin is QS-21. The ginsenoside may beginsenoside Rg1, ginsenoside Rg3, ginsenoside Rb1 or ginsenoside Re. Theplatycodin is platycodin D, platycodin D2 or a mixture thereof. Theastragaloside may be astragalin A (astragaloside IV), astragaloside I,astragaloside II, or a mixture of two or more of these saponin monomers.The notoginsenoside may be notoginsenoside R1. The ophiopogonin may beophiopogonin D. The saikosaponin may be saikosaponin a, saikosaponin d,or a mixture thereof. The cortex albiziae saponin may be cortex albiziaetotal saponins. The glycyrrhizin may be total glycyrrhizins. The panaxjaponicus saponin may be panax japonicus total saponins.

In the immunostimulatory composition according to the invention, theadjuvant comprising a saponin is an immunostimulating complex adjuvant(Iscom adjuvant).

In the immunostimulatory composition according to the present invention,the CpG oligodeoxynucleotide comprises a phosphorothioate linkage.Particularly, the CpG oligodeoxynucleotide is athio-oligodeoxynucleotide, preferably a perthio-oligodeoxynucleotide.

In the immunostimulatory composition according to the present invention,the weight ratio of the CpG oligodeoxynucleotide to the saponin is1˜40:0.1˜2, preferably 2˜40:0.1˜2, and more preferably 2:1.

In another aspect, the present invention also provides a pharmaceuticalcomposition comprising the immunostimulatory composition, and an antigenor an antigen composition.

In the pharmaceutical composition according to the present invention,the antigen or antigen composition is any one selected from the groupconsisting of human immunodeficiency virus, human herpes virus,varicella-zoster virus, human cytomegalovirus, hepatitis A, B, C or Evirus, respiratory syncytial virus, human papilloma virus, influenzavirus, Mycobacterium tuberculosis, salmonella, neisseria such asNeisseria meningitidis or Neisseria gonorrhoeae, borrelia such asborrelia recurrentis or borrelia duttonii, chlamydia such as Chlamydiatrachomatis, bordetella such as Bordetella pertussis, plasmodium such asPlasmodium falciparum, plasmodium malariae, plasmodium ovale, Plasmodiumvivax or Plasmodium knowlesi, or toxoplasma such as Toxoplasma gondii.

In the pharmaceutical composition according to the present invention,the human herpes virus is HSV1 or HSV2.

In the pharmaceutical composition according to the present invention,the antigen is a tumor antigen.

In yet another aspect, the present invention provides a vaccinecomprising the immunostimulatory composition.

The vaccine according to the present invention is a vaccine forpreventing a viral, bacterial and/or parasitic infection, or a vaccinefor treating a viral, bacterial and/or parasitic infection withimmunotherapy.

In yet another aspect, the present invention provides use of theimmunostimulatory composition in the preparation of a medicament foreliciting a cytolytic T cell response.

In some specific embodiments, the present invention provides use of theimmunostimulatory composition in the preparation of a medicament forinducing an interferon γ response in a mammal.

In some specific embodiments, the present invention provides use of theimmunostimulatory composition in the preparation of a vaccine forpreventing a viral, bacterial and/or parasitic infection.

In some specific embodiments, the present invention provides use of theimmunostimulatory composition in the preparation of a vaccine fortreating a viral, bacterial and/or parasitic infection withimmunotherapy.

In some specific embodiments, the present invention provides use of theimmunostimulatory composition in the preparation of a vaccine fortreating a tumor with immunotherapy.

The present invention also provides a method for eliciting a cytolytic Tcell response comprising administering to a subject in need thereof aneffective amount of a pharmaceutical composition comprising theimmunostimulatory composition of the present invention.

The present invention also provides a method for inducing an interferonγ response in a mammal comprising administering to a subject in needthereof an effective amount of a pharmaceutical composition comprisingthe immunostimulatory composition of the present invention.

The present invention also provides a method for preventing a viral,bacterial and/or parasitic infection comprising administering to asubject in need thereof a prophylactically effective amount of a vaccinecomprising the immunostimulatory composition of the present invention.

The present invention also provides a method for treating a viral,bacterial and/or parasitic infection with immunotherapy comprisingadministering to a subject in need thereof an effective amount of avaccine comprising the immunostimulatory composition of the presentinvention.

The present invention also provides a method for treating a tumorcomprising administering to a subject in need thereof a therapeuticallyeffective amount of a pharmaceutical composition comprising theimmunostimulatory composition of the present invention.

The immunostimulatory composition provided by the present inventionachieves an unexpected technical effect of mediating a stronger immuneresponse. The immunostimulation effect of CpG T1˜T3 alone is weaker thanthat of CpG1018, CpG7909 or CpG1826, etc. However, when they arecombined with QS-21, the immunostimulatory compositions exhibitunexpected synergistic effects, and the immune effects are significantlyenhanced.

The study of the present invention found that the hepatitis Btherapeutic vaccine containing the immunostimulatory composition couldbreak through the immune tolerance in the transgenic mice and producehigh titers of anti-HBsAg antibodies, anti-HBcAg antibodies andneutralizing antibodies. The various test results showed that thisvaccine could significantly eliminate hepatitis B virus in thetransgenic mice through multiple immunizations. At the end of theimmunization process, the HBsAb level was close to saturation, whichcould maintain a long-term stable immune effect, and the averagedecrease rate of HBsAg was maintained at about 92%. Meanwhile, theHepatitis B vaccine containing the immunostimulatory composition couldinduce production of stronger levels of HBsAg- and HBcAg-specific IFN-γ,and the immune effects were significantly better than those of eitheradjuvant alone or the combinations of the existing CPG adjuvants andQS-21.

The herpes zoster vaccine containing the immunostimulatory compositionalso demonstrates that the immunostimulatory composition has a superiorimmunostimulatory effect. The cellular immunity experiment demonstratedthat this vaccine could induce a stronger level of herpes gEprotein-specific IFN-γ, and the protein immune effect was significantlysuperior to that of a single adjuvant. The humoral immunity experimentalso demonstrated that the vaccine could generate a higher level ofherpes gE protein-specific IgG/IgG1/IgG2a antibody, and its effects weresuperior to those of a single adjuvant and significantly superior tothose of the combinations of the existing CPG adjuvants and QS-21.

In conclusion, the immunostimulatory composition provided by the presentinvention has a superior immunostimulatory effect. Compared with asingle adjuvant and combinations of the existing CPG adjuvants and QS21,CpG T1-T3 and QS-21 in the immunostimulatory composition of the presentinvention exhibit synergistic effects with high efficiency and canmediate stronger immune responses. They have significant advantages whenapplied to different antigens or antigen compositions. Therefore, as anew type of adjuvant, the immunostimulatory composition of the presentinvention has high clinical application value and broad market prospect.

BRIEF DESCRIPTION OF THE DRAWINGS

The embodiments of the present invention will be described below indetail in conjunction with the accompanying drawings, in which:

FIG. 1 shows the effects of different CPG oligodeoxynucleotides on thesecretion level of HBsAg antigen-specific IFN-γ.

FIG. 2 shows the effects of different CPG oligodeoxynucleotides on thesecretion level of HBcAg antigen-specific IFN-γ.

FIG. 3 shows the effects of different immunostimulatory compositionsaccording to the present invention on the secretion level of HBsAgantigen-specific IFN-γ.

FIG. 4 shows the effects of different immunostimulatory compositionsaccording to the present invention on the secretion level of HBcAgantigen-specific IFN-γ.

FIG. 5 shows the effects of varying dosages of the immunostimulatorycomposition according to the present invention on the secretion level ofHBsAg antigen-specific IFN-γ.

FIG. 6 shows the effects of varying dosages of the immunostimulatorycomposition according to the present invention on the secretion level ofHBcAg antigen-specific IFN-γ.

FIG. 7 shows the effects of the hepatitis B vaccines containing theimmunostimulatory composition of the present invention on the level ofHBsAg in serum.

FIG. 8 shows the effects of the hepatitis B vaccines containing theimmunostimulatory composition of the present invention on the level ofHBsAb in serum.

FIG. 9 shows the effects of the hepatitis B vaccines containing theimmunostimulatory composition of the present invention on the secretionlevel of HBsAg antigen-specific IFN-γ.

FIG. 10 shows the effects of the hepatitis B vaccines containing theimmunostimulatory composition of the present invention on the secretionlevel of HBcAg antigen-specific IFN-γ.

FIG. 11 shows the effects of the hepatitis B vaccines containing theimmunostimulatory composition of the present invention on the levels ofHBsAg antigen-specific IgG antibody and subtypes thereof in the mouseserum; wherein, Panel A: HBsAb IgG levels in the mouse serum for all thegroups; Panel B: HBsAb IgG1 levels in the mouse serum for all thegroups; Panel C: HBsAb IgG2a levels in the mouse serum for all thegroups; Panel D: the ratios of HBsAb IgG2a to IgG1 in the mouse serumfor all the groups.

FIG. 12 shows the effects of the hepatitis B vaccines containing theimmunostimulatory composition of the present invention on the levels ofHBcAg antigen-specific IgG antibody and subtypes thereof in the serum ofmice; wherein, Panel A: HBcAb IgG levels in the mouse serum for all thegroups; Panel B: HBcAb IgG1 levels in the mouse serum for all thegroups; Panel C: HBcAb IgG2a levels in the mouse serum for all thegroups; Panel D: the ratios of HBcAb IgG2a to IgG1 in the mouse serumfor all the groups.

FIG. 13 shows the effects of the herpes zoster vaccines containing theimmunostimulatory composition of the present invention on the secretionlevel of herpes gE antigen-specific IFN-γ.

FIG. 14 shows the effects of the herpes zoster vaccines containing theimmunostimulatory composition of the present invention on the levels ofantigen-specific IgG antibody and subtypes thereof in the mouse serum;wherein, Panel A: IgG levels in the mouse serum for all the groups;Panel B: IgG1 levels in the mouse serum for all the groups; Panel C:IgG2a levels in the mouse serum for all the groups; Panel D: the ratiosof IgG2a and IgG1 in the mouse serum for all the groups.

FIG. 15 shows the effects of the immunostimulatory compositionscomprising different saponins according to the present invention on thesecretion level of herpes gE antigen-specific IFN-γ.

DEFINITIONS

Unless defined otherwise, all the scientific and technical terms usedherein have the same meaning as understood by one of ordinary skill inthe art. With regard to the definitions and terms in the art, one ofskill can refer specifically to Current Protocols in Molecular Biology(Ausubel). The abbreviations for amino acid residues are standard3-letter and/or 1-letter codes used in the art to refer to one of 20common L-amino acids.

Although the present invention shows the numerical ranges andapproximations of parameters in broad scopes, the numerical values shownin the specific examples are reported as precisely as possible. All thenumerical values, however, inherently contain a certain errornecessarily resulting from the standard deviations found in theirrespective measurements. Additionally, all the ranges disclosed hereinare to be understood to encompass any and all the subranges subsumedtherein. For example, a stated range of “2 to 40” should be consideredto include any and all the subranges between (and inclusive of) theminimum value of 2 and the maximum value of 40, that is, all thesubranges beginning with a minimum value of 2 or more, e.g. 2 to 6.1,and ending with a maximum value of 40 or less, e.g. 5.5 to 40. Further,any reference referred to as “incorporated herein” is understood to beincorporated in its entirety.

It is further noted that, as used in this specification, the singularforms include the plural forms of the referents to which they refer,unless expressly and unequivocally limited to one referent. The term“or” may be used interchangeably with the term “and/or”, unless thecontext clearly dictates otherwise.

As used herein, the terms “pharmaceutical composition”, “combinationdrug”, and “drug combination” may be used interchangeably and refer to acombination of at least one drug, and optionally a pharmaceuticallyacceptable excipient or auxiliary material, which are combined togetherto achieve a certain particular purpose. In certain embodiments, thepharmaceutical composition comprises temporally and/or spatiallyseparated components, so long as they are capable of cooperating toachieve the objects of the present invention. For example, theingredients (e.g. gE protein, QS-21, and CpG oligodeoxynucleotide)contained in the pharmaceutical composition may be administered to asubject as a whole or separately. When the ingredients contained in thepharmaceutical composition are administered separately to a subject, theingredients may be administered to the subject simultaneously orsequentially.

As used herein, the term “CpG oligodeoxynucleotide” or “CpG-ODN” refersto a short single-chain synthetic DNA molecule containing one or more“CpG” unit(s), wherein C represents cytosine, G represents guanine, andp represents a phosphodiester bond. In particular, the CpGoligodeoxynucleotide is non-methylated. In some embodiments, the CpG-ODNcomprises a phosphorothioate linkage or a phosphorothioate backbone.That is to say, in some embodiments, the CpG-ODN is a phosphorothioateoligodeoxynucleotide (i.e. a thio-oligodeoxynucleotide). Preferably, allthe internucleotide linkages in the CpG-ODN are phosphorothioatelinkages, that is, the CpG-ODN is a perthio-oligodeoxynucleotide. Inother embodiments, the CpG-ODN comprises two or more copies of5′-TTCGTT-3′ motif or 5′-TCGTCGTCG-3′ motif. In particular, the CpG-ODNhas a sequence selected from: TCG TTC GTT CGT TCG TTC GTT (SEQ ID NO:6), TCG TTC GTT CGT TCG TTC GTT CUT T (SEQ ID NO: 7), or TCG TCG TCG TCGTCG TCG TCG (SEQ ID NO: 8), preferably TCG TTC GTT CGT TCG TTC GTT (SEQID NO: 6).

As used herein, “ginsenoside, platycodin, astragaloside,notoginsenoside, glycyrrhizin, cortex albiziae saponin, ophiopogonin,saikosaponin or panax japonicus saponin” refer to an active ingredientpresented in the corresponding plant. For example, ginsenoside is a kindof sterol compounds, which mainly exist in the medicinal materials ofgenus Panax and are active ingredients in ginseng. In some embodiments,the ginsenoside is preferably a monomer such as ginsenoside Rg1,ginsenoside Rg3, ginsenoside Rb1, ginsenoside Re, or a mixture of two ormore of these saponin monomers. The platycodin is preferably platycodinD, platycodin D2 or a mixture thereof. The astragaloside is preferably amonomer such as astragalin A (astragaloside IV), astragaloside I,astragaloside II, and the like, or a mixture of two or more of thesesaponin monomers. The notoginsenoside is preferably notoginsenoside R1,or the like. The ophiopogonin is preferably ophiopogonin D, or the like.The saikosaponin is preferably saikosaponin a, saikosaponin d, or amixture thereof. The cortex albiziae saponin is preferably cortexalbiziae total saponins or the like. The glycyrrhizin is preferablytotal glycyrrhizins or the like. The panax japonicus saponin ispreferably panax japonicus total saponins or the like.

As used herein, “Iscom adjuvant” is an immunostimulatory complexadjuvant, specifically ISCOM MATRIX that does not comprise an antigen,which is an adjuvan composed of a phospholipid, a saponin, andcholesterol with a cage-like structure.

As used herein, “a therapeutically and/or prophylactically effectiveamount” or “an effective amount” refers to a dosage sufficient to showits benefit to the subject to which it is administered. The actualamount administered, as well as the rate and time course ofadministration, would depend on the own conditions and severity of thesubject being treated. A prescription of treatment (e.g. determinationof dosage, etc.) is ultimately the responsibility of, and determined by,general practitioners and other physicians, often taking into accountthe disease to be treated, the conditions of the individual patient, thesite of delivery, the method of administration, and other factors knownto physicians.

As used herein, the term “mammal” refers to a human, and may also beother animals, such as wild animals (e.g. herons, storks, cranes, etc.),domestic animals (e.g. ducks, geese, etc.) or laboratory animals (e.g.chimpanzees, monkeys, rats, mice, rabbits, guinea pigs, woodchucks,ground squirrels, etc.).

In other embodiments, the composition of the present invention mayfurther comprise an additional additive, such as a pharmaceuticallyacceptable carrier or additive, particularly when presented as apharmaceutical formulation form.

The preferred pharmaceutical carrier is especially water, bufferedaqueous solutions, preferably isotonic saline solutions such as PBS(phosphate buffer), glucose, mannitol, dextrose, lactose, starch,magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol,hyaluronic acid, ethanol or polyalkylene glycols such as polypropyleneglycol, triglycerides, etc. The types of the pharmaceutical carrier useddepend inter alia on whether the composition according to the presentinvention is formulated for oral, nasal, intradermal, subcutaneous,intramuscular or intravenous administration. The composition accordingto the present invention may comprise a wetting agent, an emulsifyingagent, or buffer substance as an additive.

The pharmaceutical composition, vaccine or pharmaceutical formulationaccording to the present invention may be administered by any suitableroute, for example, oral, nasal, intradermal, subcutaneous,intramuscular or intravenous administration.

The present invention is further illustrated by the followingdescription of specific embodiments in conjunction with the accompanyingdrawings, which are not to be construed as limitation of the presentinvention, and various modifications or improvements can be made bythose skilled in the art in light of the basic concepts of the presentinvention, which are all within the scope of the present invention, aslong as they do not deviate from the basic concepts of the presentinvention.

BEST MODES FOR CARRYING OUT THE INVENTION

The present invention is illustrated below with reference to thespecific examples. Those skilled in the art will appreciate that theseexamples are merely illustrative of the present invention and notintended to limit the scope of the present invention in any way.

The experimental methods in the following examples are conventional,unless otherwise specified. The raw materials, reagent materials and thelike used in the following examples are commercially available products,unless otherwise specified.

Example 1 Preparation of Immunostimulatory Compositions and HepatitisVaccines of the Present Invention

1. HBsAg stock solution: the amino acid sequence of the HBsAg protein isshown by SEQ ID NO: 1.

The HBsAg protein was prepared from recombinant yeast cells of the HBsAggene, and the types of yeast cells include Hansenula, Saccharomycescerevisiae and Pichia, preferably Hansenula. For the specificpreparation steps, reference was made to Chinese patent applicationCN108330145A. The recombinant Hansenula cells of the HBsAg gene werecultured by fermentation and the mycelia were harvested. The myceliawere subjected to disruption treatment and purified by the steps ofsilica gel adsorption, column chromatography and TFF, etc.

2. HBcAg stock solution: the amino acid sequence of the HBcAg protein isshown by SEQ ID NO: 2.

The HBcAg protein was prepared from recombinant yeast cells of the HBcAggene, and the types of yeast cells include Hansenula, Saccharomycescerevisiae and Pichia, preferably Hansenula. For the specificpreparation steps, reference was made to Chinese patent applicationCN108047316A. The recombinant Hansenula cells of the HBcAg gene werecultured by fermentation and the mycelia were harvested. The myceliawere subjected to disruption treatment and purified by the steps ofammonium sulfate treatment, column chromatography and TFF, etc toprepare the HBcAg stock solution.

3. QS-21 was purchased from BRENNTAG, CAS. NO. A010-023.

4. Preparation method of CPG oligodeoxynucleotide raw materials:

Oligodeoxynucleotides are synthetically prepared fragments ofoligodeoxynucleotide sequence containing one or more CpG motifs. Theoligodeoxynucleotide sequences used in this example are shown in Table1:

TABLE 1 Specific sequences of CPG oligodeoxynucleotide Typing of CPG CPGOligodeoxynucleotide Oligodeoxynucleotide Sequence Type B CpG 1018TGACTGTGAACGTTCGAGATGA (SEQ ID NO: 3) CpG 7909TCGTCGTTTTGTCGTTTTGTCGTT (SEQ ID NO: 4) CpG 1826TCCATGACGTTCCTGACGTT (SEQ ID NO: 5) CpG T1TCGTTCGTTCGTTCGTTCGTT (SEQ ID NO: 6) CpG T2TCG TTC GTT CGT TCG TTC GTT CGT T (SEQ ID NO: 7) CpG T3TCG TCG TCG TCG TCG TCG TCG (SEQ ID NO: 8) CpG 684TCGACGTTCGTCGTTCGTCGTTC (SEQ ID NO: 9) CpG 1668TCC ATG ACG TTC CTG ATGCT (SEQ ID NO: 10) CpG D2TGTCGTCGTCGTTTGTCGTTTGTCGTT (SEQ ID NO: 11) Type A CpG 2216GGGGGACGATCGTCGGGGGG (SEQ ID NO: 12) ODN 2336GGGGACGACGTCGTGGGGGGG (SEQ ID NO: 13) Type C ODN 2395TCGTCGTTTCGCGCGCGCCG (SEQ ID NO: 14) ODN M362TCGTCGTTCGTTCGTCGAACGACGTTTGAT (SEQ ID NO: 15)

Specific preparation method: a conventional solid phasephosphoramidite-phosphotriester chemical synthesis method was used forthe preparation, starting from the 3′ end, i.e., 1) Deprotection: firstremoving the protecting group DMT (dimethoxytrityl) of the nucleotideconnected to CpG with trichloroacetic acid to obtain free 5′ hydroxylfor the next step of condensation reaction; 2) Activation: mixing aphosphoramidite-protected nucleotide monomer and a tetrazole activatorinto a synthesis column to form a phosphoramidite tetrazole activeintermediate, which undergoes a condensation reaction with a deprotectednucleotide on CpG; 3) Connection: when the phosphoramidite tetrazolereactive intermediate encounters a deprotected nucleotide on CpG, itwill undergo an affinity reaction with its 5′ hydroxyl, condense andremove the tetrazole, upon which the oligonucleotide chain is extendedforward by one base; 4) Oxidation: during the condensation reaction, thenucleotide monomer is connected to the oligonucleotide connected to CpGvia a phosphite bond, while the phosphite bond is unstable and prone tobe hydrolyzed by an acid or a base, upon which the phosphoramidite isoxidized into a phosphotriester with a sulphur-phosphorus double bondusing a thio-substitution reagent, thereby obtaining a stableoligonucleotide; and 5) Blocking: in order to prevent the unreacted 5′hydroxyl connected to CpG from being extended in the subsequent circularreaction after the condensation reaction, this terminal hydroxyl isoften blocked by acetylation. After the above five steps, onedeoxynucleotide is connected to the nucleotide of CpG. The abovedeprotection, activation, connection, oxidation and blocking processesare repeated to obtain a crude DNA fragment. Finally, it is subjected topost-synthesis treatments, such as cleavage, deprotection, purificationand quantification, etc.

5. The HbsAg stock solution and the HbcAg stock solution were diluted to200 μg/ml and 100 μg/ml respectively, using a PBS solution (purchasedfrom Hyclone). All the CPG raw materials were separately dissolved anddiluted to 100 μg/ml using the PBS solution for the next step.

Example 2 Screening Experiment of CPG Oligodeoxynucleotides

1. Experimental animals: C57BL/6(N) mice, male, 4 weeks old, 135 mice,Shanghai Lingchang Laboratory Animal Technology Co. Ltd.

2. Experimental grouping: see Table 2. The dosage for each injection was100 μL/mice, and Group A was the negative control (the PBS solution, 100μL/mouse).

TABLE 2 Grouping of experimental animals Component (μg/mouse) Number HBsHBc CpG CpG CpG CpG CpG CpG CpG CpG CPG CpG ODN ODN ODN Group (animals)Ag Ag T1 T2 T3 1618 7909 1826 684 1668 D2 2216 2336 2395 M362 Control 9Antigen 9 20 10 T1 9 20 10 10 T2 9 20 10 10 T3 9 20 10 10 1018 9 20 1010 7909 9 20 10 10 1826 9 20 10 10 684 9 20 10 10 1668 9 20 10 10 D2 920 10 10 2216 9 20 10 10 2336 9 20 10 10 2395 9 20 10 10 M362 9 20 10 10

3. Experimental steps: on Day 7 after the immunization of mice, thespleen lymphocytes were prepared according to a conventional method, andthe details were as follows: the spleens were taken aseptically by beingcut with sterile forceps and scissors, and placed in a 70 μm cellstrainer, which was placed in a plate containing 2 ml of pre-chilled 2%FBS (purchased from GIBCO)-PBS; the spleens were ground using a grindingrod, and the spleen cells entered the plate through the meshes to obtaina cell suspension, and then the suspension was filtered by a 40 μm cellstrainer (purchased from BD) and put into a 50 ml sterile centrifugetube by using a Pasteur pipet; it was centrifuged at 500×g at 4° C. for5 min; the supernatant was discarded, and then 2 ml of 1×erythrocytedisruption agent (purchased from BD) was added to re-suspend the cells,and the resultant was allowed to stand for 5 min at 4° C., protectedfrom light to disrupt the red blood cells; 10 ml of 2% FBS-PBS was addedto terminate the erythrocyte disruption reaction; the resultant wascentrifuged at 500×g at 4° C. for 5 min, the supernatant was discarded,and then 5 ml of 2% FBS-PBS was added to re-suspend the cells for lateruse. The spleen cells were stimulated with the stimulators,HBsAg-specific peptide library PS4 and HBcAg-specific peptide libraryPCP, respectively. An ELISPOT kit (BD) was used to detect the secretionlevels of HBsAg and HBcAg antigen-specific IFN-γ according to the kitinstructions. The spot number measured by the ELISPOT kit was read usingImmunoSPOT Series 3 Elispot analyzer (refer to Example 7 of Chinesepatent CN104043120B for the specific operation steps).

The sequences of HBsAg-specific peptide library refer to Example 7 ofChinese patent CN104043120B, and the sequences of HBcAg-specific peptidelibrary are shown by SEQ ID NO: 16˜30.

4. Experimental results: the results of ELISPOT spot are shown in FIG. 1and FIG. 2 . The results show that the CpG adjuvants of type B withdifferent sequences had different immune effects. Among them, CpG T1˜T3,CpG 1018, CpG 7909, CpG 1826 and CpG 684 as a whole were superior to theCpG adjuvants of type A and the CpG adjuvants of type C, while CpG 1618and CPG D2 had poorer immune effects, and the induced production levelsof HBsAg- and HBcAg-specific IFN-γ were all lower than those induced bythe CpG adjuvants of type A and the CpG adjuvants of type C.

Example 3 Screening Experiment of Immunostimulatory Compositions

1. Experimental animals: C57BL/6(N) mice, male, 4 weeks old, 81 mice,Shanghai Lingchang Laboratory Animal Technology Co. Ltd.

2. Experimental grouping: see Table 3. The dosage for each injection was100 μL/mice, and Group A was the negative control (the PBS solution, 100μL/mouse).

TABLE 3 Grouping of experimental animals Component (μg/mouse) Number CpGCpG CpG CpG CpG CpG CpG Group (animals) HBsAg HBcAg T1 T2 T3 1018 79091826 684 QS21 Control 9 Antigen 9 20 10 T1 9 20 10 10 5 T2 9 20 10 10 5T3 9 20 10 10 5 1018 9 20 10 10 5 7909 9 20 10 10 5 1826 9 20 10 10 5684 9 20 10 10 5

3. Experimental steps: following Example 2.

4. Experimental results: the results of ELISPOT spot are shown in FIG. 3and FIG. 4 . The results show that use of CpG T1˜T3 in combination withQS21 resulted in high-efficiency synergistic effects, and the inducedproduction levels of HBsAg- and IBcAg-specific IFN-γ were significantlyhigher than those induced by other CpG adjuvants, such as CpG 1018, CpG7909, etc., with unexpected immune effects.

Example 4 Effects of Different Amounts of Adjuvant on Immune Effect ofthe Pharmaceutical Composition

1. Experimental animals: C57BL/6(N) mice, male, 4 weeks old, 60 mice,Shanghai Lingchang Laboratory Animal Technology Co. Ltd.

2, Reagents and materials:

1) The HBsAg protein, HBcAg protein and CpG T1 were obtained fromExample 1.

2) QS-21 (CAS. NO. A010023, purchased from BRENNTAG).

3) The HBsAg stock solution and IBcAg stock solution were diluted to 200μg/ml and 100 μg/ml respectively, using a PBS solution (purchased fromHyclone); QS21 was diluted to 5 μg/ml, 50 μg/ml and 100 μg/mlrespectively, using the PBS solution; CpG T1 was dissolved and dilutedto 50 μg/ml, 100 μg/ml and 2 mg/ml respectively, using the PBS solution;and CPG 7909 was dissolved and diluted to 100 μg/mi using the PBSsolution, for the next step.

3. Experimental grouping: see Table 4. The dosage for each injection was100 μL/mouse, and Group A was the negative control (the PBS solution,100 μL/mouse).

4. Experimental steps: following Example 2.

5. Experimental results: the results of ELISPOT spot are shown in FIG. 5and FIG. 6 . The results show that the dosage changes of CpG T1 and QS21had significant effects on the vaccine compositions, and theimmunostimulatory compositions having a dosage higher than Dosage 5induced the production levels of HBsAg- and HBcAg-specific IFN-γ, whichwere significantly higher than that of CPG 7909 group. However, due tothe species difference, a further increase of adjuvant dosage did notinduce a significant increase of the effect, presumably because the micecould not accurately reflect the immune intensity of adjuvant.

Dosages 1, 2 and 4 are equivalent to CPG 7909 group in terms ofimmunostimulatory effect, but the adjuvant dosages used were lower thanthat of the equivalent CPG 7909 group, thus they also had a certainadvantage.

TABLE 4 Grouping of experimental animals Number Component (μg/mouse)Group (animals) HBsAg HBcAg CpG T1 QS21 CPG7909 Control 5 Antigen 5 2010 Dosage 1 5 20 10  5 0.5 Dosage 2 5 20 10  5 5   Dosage 3 5 20 10  510   Dosage 4 5 20 10  10 0.5 Dosage 5 5 20 10  10 5   Dosage 6 5 20 10 10 10   Dosage 7 5 20 10 200 0.5 Dosage 8 5 20 10 200 5   Dosage 9 5 2010 200 10   CPG7909 5 20 10 5   10

Example 5 Experimental Group Setting and Immunization Process ofHepatitis B Vaccines

1. Experimental animals and model establishment: C57BL/6(N) mice, male,4 weeks old, 81 mice, Shanghai Lingchang Laboratory Technology Co. Ltd.;rAAV 8-HBV adenovirus, purchased from Beijing FivePlus MolecularMedicine Institute Co. Ltd. A C57BL/6(N) mouse model infectedpersistently with rAAV 8-HBV was established by intravenously injectingrAAV 8-HBV adenovirus into the upper tail vein of C57BL/6(N) mice.

2. Reagents and materials:

1) HBsAg protein: obtained from Example 1.

2) HBcAg protein: obtained from Example 1.

3) The HBsAg stock solution, HBcAg stock solution and QS-21 were dilutedto 200 μg/ml, 100 μg/ml and 50 μg/ml respectively, using a PBS solution(purchased from Hyclone), and CpG was dissolved and diluted to 100 μg/mlusing the PBS solution, for the next step.

3. Experimental grouping: see Table 5. The dosage for each injection was100 μL/mouse, and Group A was the negative control which was injectedwith the PBS solution 100 μL/mouse.

TABLE 5 Grouping of experimental animals and injection dosage for eachgroup Number Component (μg/mouse) Group (animals) HBsAg HBcAg CpG T1 CpG7909 QS-21 A 9 B 9 10 C 9 5 D 9 10 5 E 9 20 10 F 9 20 10 10 G 9 20 10 5H 9 20 10 10 5 I 9 20 10 10 5

4. Animal immunization: all the groups were administrated byintramuscular injection once every 2 weeks and the inoculation site wasat the right rear thigh, with a total of 6 administrations at the 4th,6th, 8th, 10th, 12th and 14th week respectively after the tail veinintravenous injection of rAAV 8-HBV virus. The blood was collected onceevery 2 weeks after the start of administration, i.e., at the 4th, 6th,8th, 10th, 12th, 14th, 16th, 18th, 20th and 22th week, respectively. Allthe mice were sacrificed at the 22th week.

Example 6 Effects of Hepatitis B Vaccines on HBsAg Level in Serum

1. Detection steps for serum HBsAg: Nanjing Drum Tower Hospital wasentrusted for detection.

Using a two-step immunoassay, the binding between the sample to bedetected and the paramagnetic particles coated with the hepatitis Bsurface antibody was firstly detected; after washing, an acridiniumester-labeled hepatitis B surface antibody conjugate was added; afterwashing again, a pre-excitation solution and an excitation solution wereadded to the reaction mixture, and the relative luminescence unit (RLU)of the sample to be detected was determined; there was a positivecorrelation between the content of HBsAg in the sample and RLU, and theconcentration of HBsAg in the mouse serum sample was determined via agenerated ARCHTITECT HBsAg standard curve; finally, the concentration ofHBsAg in the mouse serum sample was 50 to 200 times of the determinedvalue.

2. Analysis of results (FIG. 7 ): Group H vaccine containing theimmunostimulator according to the present invention showed a significantdowntrend in the corresponding HBsAg level, and maintained a stablelong-lasting immune effect after the end of the immunization process(from Week 14), with a significant advantage compared to the CpG groupalone (Group F) and the QS-21 group alone (Group G). The HBsAg level inGroup H decreased from >6350 IU/ml at the onset to about 50 IU/ml. Inthis group, the HBsAg level decreased by more than 30% after the secondimmunization (Week 6) and decreased by more than 70% after the thirdimmunization (Week 8), and the average decrease rate was maintained atabout 92% after the end of immunization at Week 14. A superior immuneeffect was found. Compared with the dual adjuvant control (Group I),Group H still maintained a stable immune effect after the end ofimmunization at Week 14, and the immune level was significantly betterthan that of Group I.

Example 7 Evaluation of Humoral Immune Effects of Hepatitis B Vaccines

1. Detection steps for serum HBsAb: Nanjing Drum Tower Hospital wasentrusted for detection.

Using a two-step immunoassay, the sample to be detected was first mixedwith the paramagnetic particles coated with recombinant HBsAg (rHBsAg);after washing, an acridinium ester-labeled rHBsAg conjugate was added;after washing again, a pre-excitation solution and an excitationsolution were added to the reaction mixture, and the relativeluminescence unit (RLU) of the sample to be detected was determined;there was a positive correlation between the content of HBsAb in thesample and RLU, and the concentration of HBsAg in the mouse serum samplewas determined via a generated ARCHTITECT HBsAb standard curve; finally,the concentration of HBsAb in the mouse serum sample was 50 to 200 timesof the determined value.

2. Analysis of results (FIG. 8 ): Group H vaccine containing theimmunostimulator began to generate HBsAb (>10 mIU/ml) after the secondimmunization (Week 6), and the level of HBsAb showed a trend ofcontinuous increase as the number of immunizations increased, and thetrend of increase was significantly superior to that of the CpG groupalone (Group F) and the QS-21 group alone (Group G). Two weeks after theend of immunization (Week 16), the HBsAb level was close to saturation,reaching an HBsAb level of 4.0 logs, i.e. about 10000 mIU/ml. Theantibody level generated was also significantly superior to that of thedual adjuvant control (Group I).

Example 8 Evaluation of Cellular Immune Effects of Hepatitis B Vaccines

1. Detection steps: following Example 2.

2. Evaluation indicators: if the spot number of control well ≤5 SFC andthe spot number of sample well ≥10 SFC, it will be determined aspositive; if 5 SFC<the spot number of control well ≤10 SFC, and the spotnumber of sample well/the spot number of control well ≥2, it will bedetermined as positive; and if the spot number of control well >10 SFC,and the spot number of sample well/the spot number of control well ≥3,it will be determined as positive.

3. Experimental results:

TABLE 6 Positive conversion rates of HBsAg- and HBcAg-specific IFN-γsecreted by spleen cells Number Positive conversion rate (%) Group(animals) HBsAg HBcAg A 9 11.1  11.1  B 9 0   11.1  C 8 0   0   D 812.5  12.5  E 9 37.5  12.5  F 9 100    100    G 8 100    100    H 8100    100    I 8 100    100   

Detection results of cellular immune level: the results of ELISPOT spotare shown in FIGS. 9 and 10 , and the analysis results show that thepositive conversion rates of HBsAg-specific IFN-γ were 100% and thepositive conversion rates of HBcAg-specific IFN-γ were 100% for GroupsF-I. Group H vaccine containing the immunostimulator could induce higherproduction levels of HBsAg- and HBcAg-specific IFN-γ, greater than 2350SFC/10⁶ spleen cells and greater than 1250 SFC/10⁶ spleen cells,respectively, with significant differences compared to the CpG alonegroup (Group F) and the QS-21 group alone (Group G). The productionlevels of HBsAg- and HBcAg-specific IFN-v induced by the dual adjuvantcontrol (Group I) were about 1630 SFC/10⁶ spleen cells and 750 SFC/10⁶spleen cells, which were significantly lower than those induced by GroupH.

Example 9 Detection of HBsAE- and HBeAg-Specific Antibodies in SerumInduced by Pharmaceutical Compositions

1. Detection steps: a 96-well ELISA plate was coated with the purifiedHBsAg and HBcAg to form solid phase antigens. After blocking treatment,the serum to be detected was diluted serially at a certain initialdilution, and multiple dilutions were set. The serially diluted serumsamples were added to the 96-well ELISA plate, and then bond toHRP-labeled anti-IgG/IgG 1/IgG 2a antibody to form antigen-antibody(serum)-enzyme labeled antibody complexes. Finally, the substrate TMBwas added for color development, and the absorbance (OD value) at 450 nmwas measured with a microplate reader. The shade of developed color waspositively correlated with the levels of HBsAg- and HBcAg-specificantibodies IgG/IgG 1/IgG 2a in the samples to be detected. Thedetermination of antibody titers was performed by fitting therelationship curve of “absorbance OD value-dilution factor of serumsample (Log)”.

2. Analysis of results:

1) Detection results of HBsAb IgG antibody and subtypes thereof inserum.

An ELISA method was used to detect the levels of HBsAb IgG antibody andsubtype thereof in the mouse serum of each group at different time. Asshown in FIG. 11 , Group H vaccine containing the immunostimulatorgenerated a higher titer of anti-HBsAg-specific IgG/IgG 1/IgG 2aantibody, and with the increase of immunization number, the antibodylevels continued to increase, and at the sixth immunization (Week 14),the antibody levels approached saturation, and the specific antibodytiters could reach more than 5.4 log. No specific antibodies weredetected in Groups A-D. Although Groups E-G generated a certain level ofHBsAg-specific IgG/IgG 1/IgG 2a antibody, the level of antibody wassignificantly lower than that in Group H. The levels ofanti-HBsAg-specific IgG antibody and IgG 2a antibody generated in thedual adjuvant control (Group I) were significantly lower than those inGroup H.

2) Detection results of HBcAb IgG antibody and subtypes thereof inserum.

An ELISA method was used to detect the levels of HBcAb IgG antibody andsubtype thereof in the mouse serum of each group at different time. Asshown in FIG. 12 , Group H vaccine containing the immunostimulatorgenerated a higher titer of anti-HBcAg-specific IgG/IgG 1/IgG 2aantibody, and with the increase of immunization number, the antibodylevel continued to increase, and at the sixth immunization (Week 14),the antibody level approached saturation, and the specific antibodytiter could reach more than 4.8 log. No specific antibodies weredetected in Groups A-D. Although Groups E-G generated a certain level ofHBcAg-specific IgG/IgG 1/IgG 2a antibody, the level of antibody wassignificantly lower than that in Group H. And Group H is more inclinedto Th1 pathway, and the specific antibody IgG 2a appeared a significantupward trend as shown in Figure D, reflecting that the vaccine of GroupH could promote the subtype conversion of anti-IBcAg antibody, and theconversion efficiency was significantly higher than that of the dualadjuvant control (Group I).

Example 10 Experimental Group Setting for Herpes Zoster Vaccines

1. Experimental animals and model establishment:

C57BL/6(N) mice, female, 5 weeks old, 48 mice, purchased from ShanghaiSLRC Laboratory Animal Co., Ltd.

2. Reagents and materials:

1) Herpes gE protein: the amino acid sequence is shown by SEQ ID NO: 31.

For the preparation steps, reference was made to the report of areference, Thomsson E., Persson L. et al. “Journal of VirologicalMethods”, 2011, Vol. 175, No. 1, pp. 53-59, and the specific steps wereas follows: according to the target protein sequence, the nucleic acidsequence was optimized so that its codons accorded with a mammalianexpression system, and the target gene was synthesized. The synthesizedtarget gene was ligated with pcDNA3.1(+) plasmid by a way of enzymedigestion and ligation, and transformed into Top 10 competence. Thepositive monoclones were picked up and verified by sequencing. Themonoclonal bacteria were amplified massively, and a large number ofplasmids suitable for cell transfection were extracted using anendotoxin-free plasmid extraction kit. Suspending CHO cells weretransfected with the plasmids by a way of transient transfection. Whenthe viability of CHO cells was less than 70% or the fermentation timewas more than 7 days, the supernatant of fermentation broth wascollected by centrifugation at 5000 rpm at 4° C. for 30 min. Thefermentation broth was dialyzed into a solution containing 50 mMTris-HCl, 500 mM NaCl and 20 mM imidazole with a dialysis ratio of 1:100in a chromatography cabinet at 4° C., once every 4 h, for a total of 3times. The collected samples were purified through a nickel column, andan SDS-PAGE detection was performed on the collected samplescorresponding to the target protein peak. The purified solutions havinga higher purity were combined and dialyzed with a solution containing 20mM phosphate and 150 mM NaCl in a chromatography cabinet at 4° C. for 24h with a dialysis ratio of 1:100, and the dialysis solution was changedevery 8 h. The samples were filtered through a 0.22 μm sterile filtermembrane and stored in a refrigerator at 4° C. for later use.

It was required that the prepared herpes gE protein stock solution had apurity of greater than 95%, a protein content of not less than 200μg/ml, and an endotoxin level of not higher than 0.1 Eu/μg.

2) The herpes gE stock solution was diluted to 50 μg/ml and 10 μg/mlusing a PBS solution (purchased from Hyclone), respectively; QS-21 wasdiluted to 50 μg/ml and 10 μg/ml using the PBS solution, respectively;CpG was diluted to 100 μg/ml and 20 μg/ml using the PBS solution,respectively; and CpG7909 was diluted to 100 μg/ml and 20 μg/ml usingthe PBS solution, respectively.

3. Experimental grouping: see Table 7. The dosage for each injection was100 μL/mouse, and Group A was the negative control which was injectedwith the PBS solution at 100 μL/mice.

TABLE 7 Grouping of experimental animals and injection amount for eachgroup Component μg/mouse Number Herpes gE Group (animals) protein CpG T1CpG7909 QS-21 A 6 B 6 5 C 6 5 10 D 6 5 5 E 6 5 10 5 F 6 5 10 5 G 6 1  21 H 6 1  2 1

4. Animal immunity: all the groups were administrated by intramuscularinjection once every 2 weeks and the inoculation site was at the rightrear thigh. They were administered twice continuously, that is, byinjection at Weeks 0 and 2, respectively. All the mice were sacrificedat Week 4.

Example 11 Verification of Cellular Immunity Efficacy of Herpes ZosterVaccines

1. The detection steps and evaluation indicators were the same as thosein Example 2, and the sequences of gE-specific peptide library are shownby SEQ ID NO. 32-46.

2. Experimental results: the levels of spot number of T-lymphocytesecreting gE-specific IFN-γ in the spleen cells of mice in each groupare shown in FIG. 13 , and the positive conversion rate results ofgE-specific IFN-γ are shown in Table 8. The results show that the levelsof spot number of T-lymphocyte secreting gE-specific IFN-γ in the spleencells corresponding to Groups E and F with a higher immune dosage (>4000SFC/10⁶ spleen cells) were significantly higher than those of Groups Gand H with a lower immune dosage. Among them, the levels of spot numberof T-lymphocyte secreting gE-specific IFN-γ in the spleen cellscorresponding to Groups E and G (CpG T1+QS-21) were higher than those ofGroups F and H (CpG 7909+QS-21) with the same dosage. The positiveconversion rates of IFN-γ for Groups E-H were 100%.

TABLE 8 Positive conversion rates of SgE-specific IFN-γ secreted byspleen cells Group A B C D E F G H Number 1/6 0/6 4/6 5/6 6/6 6/6 6/66/6 of positive conversion/ mice Positive 16.7 0 66.7 83.3 100 100 100100 conversion rate/%

Example 12 Verification of Humoral Immunity Efficacy of Herpes ZosterVaccines

1. Detection steps: on Day 28 after the immunization, the blood wascollected and the serum was separated (the whole blood was placed in anincubator with a constant temperature of 37° C. for 40 min andcentrifuged at 12000 rpm at 4° C. for 10 min; the supernatant was suckedand cryopreserved at −20° C. for later use). An ELISA kit (ShanghaiKehua) was used to detect the positive conversion rates of herpes gEprotein-specific antibodies according to the kit instructions. For thedetection, a blank control, a negative control and the samples to bedetected were set, so that each of them had two parallel wells, whereinthe negative control was negative mouse serum; except for the blankcontrol, the negative control or the sample to be detected was added toeach well followed by an enzyme conjugate. After mixing and sealing theplates, the plates were incubated at 37° C. for 30 min. Each well waswashed with a washing solution and added with developer solution A anddeveloper solution B. After mixing and sealing the plates, the plateswere incubated at 37° C. for 15 min. A termination solution was addedinto each well and mixed evenly. The OD value of each well at awavelength of 450 nm was read using a microplate reader.

2. Experimental results: the levels of antigen-specific IgG antibody andsubtypes thereof in the mouse serum detected by ELISA are shown in FIG.14 . The results show that the immune effect of Group B containing theimmunostimulator of the present invention was significantly superior tothat of the group CpG alone (group C), the group QS-21 alone (Group D)and the dual adjuvant control (Group F). Moreover, the correspondinglevels of IgG and IgG 2a antibodies were significantly different fromthose of the two groups. That is, addition of CpG to QS-21 couldincrease the corresponding humoral immune level.

Example 13 Effects of Different Saponins on Efficacy of the RecombinantHerpes Zoster Vaccine Composition

1. Experimental animals and model establishment:

C57BL/6(N) mice, female, 5 weeks old, 48 mice, purchased from ShanghaiSLRC Laboratory Animal Co. Ltd.

2. Reagents and materials:

1) The herpes gE protein was obtained from Example 10, and CpG T1 andCpG 7909 were prepared from Example 1.

2) QS-21 (CAS: NO. A010-023, purchased from BRENNTAG); ginsenoside Rg1(CAS: 22427-39-0, purchased from Nanjing Spring & Autumn BiologicalEngineering Co. Ltd.); astragalin A (CAS: 84687-43-4, purchased fromNanjing Spring & Autumn Biological Engineering Co. Ltd.); platycodin D(CAS: 58479-68-8, purchased from Hubei Yunmei Technology Co. Ltd.);Iscom adjuvant (purchased from Shanghai Xiyuan Biotechnology Co. Ltd.).

3) The herpes gE stock solution was diluted to 50 μg/mL using a PBSsolution (purchased from Hyclone). All the saponins were separatelydiluted to 50 μg/mL using the PBS solution. CpG T1 and CpG 7909 weredissolved and diluted to 100 μg/mL respectively, using the PBS solution,for the next step.

3. Experimental grouping:

See Table 9. The dosage for each injection was 100 μL/mice, and thecontrol group was injected with the PBS solution at 100 μL/mouse.

4. Experimental steps: following Example 2.

5. Experimental results:

The results of ELISPOT spot are shown in FIG. 15 . The results show thatuse of CpG T1 in combination with various saponins resulted in ahigh-efficiency synergistic effect, and the induced production levels ofgE-specific IFN-γ were significantly higher than those by thecompositions of other CpGs and saponins, wherein QS21 had the besteffect.

TABLE 9 Grouping of experimental animals Component (μg/mouse) Number CpGGinsenoside Platycodin Astragaloside Iscom CpG Group (animals) gE T1QS-21 Rg1 D IV adjuvant 7909 Control 6 Antigen 6 5 QS-21 6 5 10 5Ginsenoside 6 5 10 5 Rg1 Platycodin D 6 5 10 5 Astragalin A 6 5 10 5Iscom 6 5 10 5 7909 6 5 5 10

In conclusion, the immune composition provided by the present inventionhas a superior immunostimulatory effect. Compared with a single adjuvantand the combinations of other CPG adjuvants and QS21, CpG T1˜T3 andQS-21 show a high-efficiency synergistic effect and can mediate astronger immune response. They have significant advantages when appliedto different antigens or antigen compositions. Therefore, as a new typeof adjuvant, this immune composition has a high clinical applicationvalue and broad market prospect.

Although the present invention has been described in detail above, thoseskilled in the art will appreciate that various modifications andvariations can be made to the present invention without departing fromthe spirit and scope of the present invention. The right scope of thepresent invention is not to be limited by the foregoing detaileddescription, and the modifications and variations are intended to fallwithin the scope of the claims. While only examples of specificembodiments of the present invention have been described above, it willbe appreciated by those skilled in the art that the foregoing isillustrative only and that the protection scope of the present inventionis to be defined by the appended claims. Various variations andmodifications can be made by those skilled in the art in the embodimentswithout departing from the principle and essence of the presentinvention, but such variations or modifications should all fall withinthe protection scope of the present invention.

1. An immunostimulatory composition comprising a saponin and a CpGoligodeoxynucleotide, or consisting of a adjuvant comprising a saponinand a CpG oligodeoxynucleotide, wherein the sequence of the CpGoligodeoxynucleotide has two or more copies of 5′-TTCGTT-3′ motif or5′-TCGTCGTCG-3′ motif.
 2. The immunostimulatory composition according toclaim 1, wherein the sequence of the CpG oligodeoxynucleotide is any oneselected from: CpG T1: TCG TTC GTT COT TCG TTC GTT (SEQ ID NO: 6); CpGT2: TCG TTC GTT CGT TCG TTC GTT CGT T (SEQ ID NO: 7); and CpG T3: TCGTCG TCG TCG TCG TCG TCG (SEQ ID NO: 8); preferably, the sequence of theCpG oligodeoxynucleotide is CpG T1: TCG TTC GTT CGT TCG TTC GTT (SEQ IDNO: 6).
 3. The immunostimulatory composition according to claim 1 or 2,wherein the saponin is one or more selected from the group consisting ofquillaja saponin, ginsenoside, platycodin, astragaloside,notoginsenoside, glycyrrhizin, cortex albiziae saponin, ophiopogonin,saikosaponin or panax japonicus saponin.
 4. The immunostimulatorycomposition according to claim 3, wherein the quillaja saponin is QS-7,QS-17, QS-18 or QS-21, preferably QS-21; the ginsenoside is ginsenosideRg1, ginsenoside Rg3, ginsenoside Rb1 or ginsenoside Re; the platycodinis platycodin D, platycodin D2 or a mixture thereof; the astragalosideis astragalin A, astragaloside I, astragaloside II, or a mixture of twoor more of these saponin monomers; the notoginsenoside isnotoginsenoside R1; the ophiopogonin is ophiopogonin D; the saikosaponinis saikosaponin a, saikosaponin d or a mixture thereof; the cortexalbiziae saponin is cortex albiziae total saponins; the glycyrrhizin istotal glycyrrhizins; and the panax japonicus saponin is panax japonicustotal saponins.
 5. The immunostimulatory composition according to anyone of claims 1 to 4, wherein the adjuvant comprising a saponin is Iscomadjuvant.
 6. The immunostimulatory composition according to any one ofclaims 1 to 5, wherein the CpG oligodeoxynucleotide comprises aphosphorothioate linkage.
 7. The immunostimulatory composition accordingto claim 5, wherein the CpG oligodeoxynucleotide is aperthio-oligodeoxynucleotide.
 8. The immunostimulatory compositionaccording to any one of claims 1 to 7, wherein the weight ratio of theCpG oligodeoxynucleotide to the saponin is 1˜40:0.1˜2, preferably2˜40:0.1˜2, more preferably 2:1.
 9. A pharmaceutical compositioncomprising the immunostimulatory composition of any one of claims 1 to8, and an antigen or antigen composition.
 10. The pharmaceuticalcomposition according to claim 9, wherein the antigen or antigencomposition is any one selected from the group consisting of humanimmunodeficiency virus, human herpes virus, varicella-zoster virus,human cytomegalovirus, hepatitis A, B, C or E virus, respiratorysyncytial virus, human papilloma virus, influenza virus, Mycobacteriumtuberculosis, salmonella, neisseria such as Neisseria meningitidis orNeisseria gonorrhoeae, borrelia such as borrelia recurrentis or borreliaduttonii, chlamydia such as Chlamydia trachomatis, bordetella such asBordetella pertussis, plasmodium such as Plasmodium falciparum,plasmodium malariae, plasmodium ovale, Plasmodium vivax or Plasmodiumknowlesi, or toxoplasma such as Toxoplasma gondii.
 11. Thepharmaceutical composition of claim 10, wherein the human herpes virusis HSV1 or HSV2.
 12. The pharmaceutical composition of claim 9, whereinthe antigen is a tumor antigen.
 13. A vaccine comprising theimmunostimulatory composition of any one of claims 1 to 8; preferably,the vaccine is a vaccine for preventing a viral, bacterial and/orparasitic infection, or the vaccine is a vaccine for treating a viral,bacterial and/or parasitic infection with immunotherapy.
 14. Use of theimmunostimulatory composition of any one of claims 1 to 8 in thepreparation of a medicament for eliciting a cytolytic T cell response.15. Use of the immunostimulatory composition of any one of claims 1 to 8in the preparation of a medicament for inducing an interferon γ responsein a mammal.
 16. Use of the immunostimulatory composition of any one ofclaims 1 to 8 in the preparation of a vaccine for preventing a viral,bacterial and/or parasitic infection.
 17. Use of the immunostimulatorycomposition of any one of claims 1 to 8 in the preparation of a vaccinefor treating a viral, bacterial and/or parasitic infection withimmunotherapy.
 18. Use of the immunostimulatory composition of any oneof claims 1 to 8 in the preparation of a vaccine for the treating atumor with immunotherapy.
 19. A method for eliciting a cytolytic T cellresponse comprising administering to a subject in need thereof aneffective amount of the pharmaceutical composition of any one of claims9 to
 12. 20. A method for inducing an interferon γ response in a mammalcomprising administering to a subject in need thereof an effectiveamount of the pharmaceutical composition of any one of claims 9 to 12.21. A method for preventing a viral, bacterial and/or parasiticinfection comprising administering to a subject in need thereof aprophylactically effective amount of the vaccine of claim
 13. 22. Amethod for treating a viral, bacterial and/or parasitic infection withimmunotherapy comprising administering to a subject in need thereof atherapeutically effective amount of the vaccine of claim
 13. 23. Amethod for treating a tumor comprising administering to a subject inneed thereof a therapeutically effective amount of the pharmaceuticalcomposition of any one of claims 9 to 12.